Cotton (Gossypium spp.) is the world's most important natural textile fiber and is also a significant oilseed crop. Cotton production provides income for approximately 100 million families, and approximately 150 countries are involved in cotton import and export. Its economic impact is estimated to be approximately $500 billion/year worldwide. World consumption of cotton fiber is approximately 115 million bales or approximately 27 million metric tons per year (National Cotton Council, on the world wide web at cotton.org, 2006). The genus Gossypium is relatively complex and includes approximately 45 diploid (2n=2x=26) and five tetraploid (2n=4x=52) species, all exhibiting disomic patterns of inheritance. Diploid species (2n=26) fall into eight genomic groups (A-G, and K). The African Glade, comprising the A, B, E, and F genomes, occurs naturally in Africa and Asia, while the D genome Glade is indigenous to the Americas. A third diploid Glade, including C, G, and K, is found in Australia. All 52 chromosome species, including Gossypium hirsutum and Gossypium barbadense, are classic natural allotetraploids that arose in the New World from interspecific hybridization between an A genome-like ancestral African species and a D genome-like American species. The closest extant relatives of the original tetraploid progenitors are the A genome species Gossypium herbaceum (A1) and Gossypium arboreum (A2) and the D genome species Gossypium raimondii (D5) ‘Ulbrich’. Polyploidization is estimated to have occurred 1 to 2 million years ago, giving rise to five extant allotetraploid species. Interestingly, the A genome species produce spinnable fiber and are cultivated on a limited scale, whereas the D genome species do not. More than 95% of the annual cotton crop worldwide is G. hirsutum, Upland or American cotton, and the extra-long staple or Pima cotton (G. barbadense) accounts for less than 2% (National Cotton Council, on the world wide web at cotton.org, 2006).
Each cotton fiber is a differentiated single epidermal cell of the ovule. Approximately half a million fibers are produced per cotton boll, some forming fuzz and some forming lint. Initiation of an epidermal cell into fiber requires a change in cell fate, which is a fundamental biological process involving genetic, physiological and developmental “switches”. Genetic mutations, polyploidy, pollination/fertilization, and hormonal regulation can affect the number of cells developing into fibers or alter fiber cell properties (fuzz vs. lint). However, it is unclear how these factors control gene expression changes that orchestrate the pattern and tempo in early stages of fiber development.
In contrast, the morphological development of cotton fibers is well documented in the art. Cotton fibers undergo four overlapping developmental stages: fiber cell initiation, elongation, secondary wall biosynthesis, and maturation. Fiber initiation is a rapid process. The white fluffy fibers begin to develop immediately after anthesis and continue up to 3 days post-anthesis (DPA), which is followed by fiber cell elongation (until 20 DPA). Secondary wall biosynthesis initiates around 15 dpa and continues to 45 DPA, followed by a maturation process at 45-60 DPA. Cotton fibers are derived from ovular epidermal cells (maternal tissues). However, only ˜25-30% of the epidermal cells differentiate into the commercially important lint fibers. The majority of cells does not differentiate into fibers or develop into short fibers or fuzz. For the cells committed to fiber development, cell initiation and elongation are nearly synchronous on each ovule, indicating that changes in gene expression are orchestrated during fiber differentiation and development through intercellular signaling and/or timing mechanisms.
In many instances, it may be advantageous to preferentially, selectively or specifically express genes in fiber developing cells or fibers. Such expression can influence the fiber development and result in longer or stronger fibers. WO 98/00549 describes the expression of cellulose synthase gene in fiber-developing cells. WO 08/012058 and WO02/45485 describe expression of sucrose synthase genes in cotton fiber developing cells. WO05/017157 describes reduction of the expression of β-1,3-glucanase in fiber-developing cells.
Cotton fiber consists of cellulose, a natural polymer composed of many molecules of the sugar glucose. Its unique structure is ideally suited for textile production. Each fiber is basically a hollow tube a few centimeters in length that, when spun and woven, provides the very special characteristic “feel” of cotton. Natural cellulose containing fibers, however, do not possess the chemical versatility of synthetic fibers, due to the relative inert nature of the cellulose consisting of β-1-4 linked glucose monomers.
WO06/136351, WO11/089021 and WO12/048807 all describe methods and means for altering cell wall reactivity in fibers of fiber producing plants such as cotton, by inclusion of positively charged oligosaccharides or polysaccharides into the cell wall. To this end, N-acetylglucosamine transferases, including chitin synthases, are expressed in fibers of the plants, and optionally also glutamine:fructose-6-phosphate amidotransferase. Although, chitin could be efficiently produced in cotton plant cell walls, it was also observed that the transgenic plants usually exhibited some reduced growth which may be attributed to a negative effect of the expression of the transgene in cotton outside of the cotton fibers.
It would thus be advantageous to be able to increase the tissue-selectivity of the expression of transgenes in fiber-producing plants, in particular to be able to increase the selectivity of expression in fibers and/or fiber-developing cells, while expression in other cells of the fiber-producing plant is substantially reduced or abolished. This could be conveniently achieved by including into the recombinant DNA construct of interest, a target site for a microRNA, preferably an endogenous miRNA, differentially expressed between fibers and the rest of the plant, preferably absent in fibers and fiber-developing cells and ubiquitously expressed in all other parts of the plant, so that expression of the miRNA in cells other than fiber or fiber-developing cells directs the post-transcriptional cleavage of any messenger RNA originating by adventitious transcription of the recombinant DNA construct of interest incorporating the miRNA target site, in cells outside the fiber developing cells or fibers. A mirroring selectivity profile (ie. expression limited to cells outside of the fiber developing cells) can also be envisaged.
Incorporation of miRNA target sequences in chimeric constructs has also been described as a trigger for the production of so-called tasiRNAs (trans-acting siRNAs) see e.g. WO 2006/138638 or WO2007/039454.
WO2006/111512 describes improved methods controlling gene expression in the field of genetics, especially plant genetics, and provides agents capable of controlling gene expression. The document specifically provides sequences of naturally occurring, tissue-specifically expressed microRNAs. The patent application further provides for transgenic expression constructs comprising sequences interacting with said microRNAs. By incorporation of the microRNA encoding sequence the expression from said expression construct is specifically silenced in the tissue where the naturally occurring microRNA is naturally expressed. Thereby the expression profile resulting from the promoter is modulated and leakiness is reduced. The document further provides for a method for modulating transgenic expression by incorporating sequences encoding said microRNAs into transgenic expression constructs. The compositions and methods of the invention can be used to enhance performance of agricultural relevant crops and for therapy, prophylaxis, research and diagnostics in diseases and disorders, which afflict mammalian species.
WO2007/047016 describes methods for producing non-natural hybrid seed. Also disclosed are miRNAs and miRNA recognition sites useful for conferring inducible sterility on a crop plant, and recombinant DNA construct including such exogenous miRNA recognition sites.
WO2008/133643 discloses novel microRNAs and their precursors, and recombinant DNA constructs including such novel miRNAs, miRNA precursors, miRNA promoters, and miRNA recognition sites corresponding to the miRNAs. Included are novel miRNA and miRNA precursors that exhibit nutrient-responsive expression. Also disclosed are miRNA decoy sequences. Further provided are non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct as described in the patent application and methods of controlling gene expression using such recombinant DNA constructs.
WO2009/003078 provides molecular constructs and methods for the temporally specific control of gene expression in plants or in plant pests or pathogens. More specifically, this patent application provides plant miRNA genes having novel circadian expression patterns that are useful for designing recombinant DNA constructs for temporally specific expression of at least one gene. Also provided are non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct of this invention.
Kwak et al. (BMC Genomics 2009, 10:457 entitled “Enrichment of a set of microRNAs during the cotton fiber development” describes a deep sequencing approach to investigate global expression and complexity of small RNAs during cotton fiber initiation and development. Two small RNA libraries were prepared and analyzed from wild-type and fuzz/lintless cotton ovules. The study demonstrated significant differences in expression abundance of miRNAs between the wild-type and mutant and suggests that these differentially expressed miRNAs potentially regulate transcripts distinctly involved in cotton fiber development.
Wang et al. (Molecular Plant 2012, Volume 5 Number 4, pages 889-900 entitled “A comparative miRNAome analysis reveals seven fiber initiation-related and 36 novel miRNAs in developing cotton ovules” describes high throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fibreless mutant during fiber initiation.
The art does remains deficient in describing microRNA molecules with the appropriate expression profile, in particular microRNAs which are expressed and processed in some or all parts of fiber producing plants, particularly cotton plants, except for fibers or fiber developing cells, and whose target sequences could be used to increase the specificity of expression of transgenes (including N-acetylglucosamine transferase and/or glutamine:fructose-6-phosphate amidotransferase) in fibers or fiber-developing cells (compared to the rest of the plant). These and other problems are solved as described hereinafter in the different embodiments, examples and claims.